Cytotherapy, 2014; 16: 90e100
Third-party umbilical cord bloodederived regulatory T cells prevent xenogenic graft-versus-host disease
SIMRIT PARMAR1, XIAOYING LIU1, SHAWNDEEP S. TUNG1, SIMON N. ROBINSON1, GABRIEL RODRIGUEZ1, LAURENCE J.N. COOPER2, HUI YANG3, NINA SHAH1, HONG YANG1, MARINA KONOPLEVA3, JEFFERY J MOLLDREM1, GUILLERMO GARCIA-MANERO3, AMER NAJJAR4, ERIC YVON1, IAN MCNIECE1, KATY REZVANI1, BARBARA SAVOLDO6, CATHERINE M. BOLLARD5 & ELIZABETH J. SHPALL1 1
Department of Stem Cell Transplantation and Cellular Therapy, 2Division of Pediatrics, 3Department of Leukemia, and 4Department of Experimental Diagnostic Imaging, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas, USA, and 5Department of Pediatrics, Medicine and Immunology and 6Department of Pediatrics and Immunology, Baylor College of Medicine, Houston, Texas, USA Abstract Background aims. Naturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graftversus-host disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1e5% of total nucleated cells in cord blood (CB) (90% (0.5e3.0 106 cells) (Figure 2A). The “negative” fraction containing CD25 cells was defined as “Tcon.” For the purpose of expansion, the whole of the positive fraction (Treg) was used. For Tcon, where appropriate, only a part (5 106 cells) of the negative fraction was cultured. Ex vivo culture was initiated with CD25þ cells at a concentration of 1 106 cells/mL in medium supplemented (as previously described) with IL-2 at 200 IU/mL, in the continued presence of CD3/28 beads, with an initial ratio of three Dynabeads:one cell. The cell numbers were assessed on an everyother-day basis and maintained at a concentration of
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Figure 2. Expanded CB Treg exert suppressive function. (A) Surface phenotype of CD25 positively and negatively selected CB T cells. Freshly isolated, CB-derived CD25þ and CD25neg fractions were analyzed at day 0, before expansion. (B) Treg phenotype. Ex vivoeexpanded CB cells after 14 days of culture were confirmed as CD4þ25þFOXP3þ127lo. (C) Expanded Treg show FOXP3 demethylation. Bisulfite sequencing in ex vivoeexpanded UCB-derived Treg showed significant DNA demethylation of the FOXP3 gene locus at amplicons 7, 9, 10 and 11 (11) when compared with Tcon. X-axis denotes the CpG island locus; Y-axis denotes percent methylation. (n ¼ 10). (D) Distribution of TCR repertoire is preserved in ex vivoeexpanded Treg: TCR repertoire remains preserved after expansion. Representative TCR repertoire distribution at day 14 of ex vivo expansion is shown. (E) Ex vivoeexpanded, CB-derived Treg significantly suppress immune response in allogeneic MLR. PBMCs from two donors were cultured together to generate MLR (D1 þ D2). Addition of Treg to the donor mixture (D1 þ D2) at a ratio of 1:1 significantly suppressed MLR. Y-axis denotes counts per minute (mean standard error of the mean, n ¼ 10). (F) Additive effect of p38MAPK inhibition. Pharmacologic inhibition of p38 MAP kinase (SB203580, 1 mmol/L) led to additive effect on CB Tregemediated MLR suppression (mean standard error of the mean, n ¼ 2).
Third-party umbilical CBederived regulatory T cells prevent xenogenic GvHD
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Figure 3. Treg infusion prevents xenogeneic GvHD. (A) Circulating human lymphocytes. No effect of addition of CB Treg on day e1 was demonstrated on the circulating human lymphocytes in the PB of the xenogenic mouse model. (B) Condition of coat. Whereas PBMC recipients (left) show extensive loss of hair and skin erythema, recipients of Treg and PBMC (right) show an intact coat of fur. (C) Weight loss. Significantly greater weight loss was detected in the xenogeneic GvHD model in PBMC recipients after as little as 14 days after transplant when compared with recipients of Treg and PBMC (P ¼ 0.002; t test; n ¼ 20 mice in each arm). (D) GvHD score. Recipients of prophylactic Treg (107) demonstrated consistently lower GvHD scores on the basis of the Ferrara GvHD scale (P < 0.001; t test). An assessment of GvHD scoring was performed every 48e72 h (n ¼ 20 mice/group). (E) Histopathologic analysis. Formalin-fixed tissues were embedded in paraffin, and sections were stained with hematoxylin and eosin. Microscopic sections of lung from recipients of PBMC alone (left panel) show areas of evidence of GvHD in the form of apoptotic bodies, lymphocytic infiltration and loss of architecture in small intestine, liver and lung. Bone marrow aplasia and lymphocytic infiltration into the spleen was present. In contrast, microscopic evaluation of small intestine, liver and lung tissue from recipients of Treg and PBMC revealed preserved tissue histology. Preserved lymphoid follicles in spleen and hematopoiesis in bone marrow were observed. (F) Treg suppress levels of inflammatory cytokines in the xenogeneic GvHD mouse model. Measurements of circulating serum cytokines at day 14 showed a consistent decrease in the pro-inflammatory cytokine levels, including IL-6, interferon-g, interferon inducible protein-10, IL-5, macrophage inflammatory protein-1b and tumor necrosis factor-a in the Treg and PBMC recipients as compared with the PBMC-only recipients (n ¼ 5 mice/group; mean standard error of the mean). The unit of the Yaxis is pg/mL. (G) Overall survival. At a median follow-up of 30 days after PBMC infusion, the overall survival of PBMC recipients that also received Treg were >80% as compared with 90% of the final Treg product at 14 days of ex vivo culture were CD4þ25þFOXP3þ127lo (Figure 2B). By comparison,
Third-party umbilical cord bloodederived regulatory T cells prevent xenogenic graft-versus-host disease
SIMRIT PARMAR1, XIAOYING LIU1, SHAWNDEEP S. TUNG1, SIMON N. ROBINSON1, GABRIEL RODRIGUEZ1, LAURENCE J.N. COOPER2, HUI YANG3, NINA SHAH1, HONG YANG1, MARINA KONOPLEVA3, JEFFERY J MOLLDREM1, GUILLERMO GARCIA-MANERO3, AMER NAJJAR4, ERIC YVON1, IAN MCNIECE1, KATY REZVANI1, BARBARA SAVOLDO6, CATHERINE M. BOLLARD5 & ELIZABETH J. SHPALL1 1
Department of Stem Cell Transplantation and Cellular Therapy, 2Division of Pediatrics, 3Department of Leukemia, and 4Department of Experimental Diagnostic Imaging, The University of Texas, M.D. Anderson Cancer Center, Houston, Texas, USA, and 5Department of Pediatrics, Medicine and Immunology and 6Department of Pediatrics and Immunology, Baylor College of Medicine, Houston, Texas, USA Abstract Background aims. Naturally occurring regulatory T cells (Treg) are emerging as a promising approach for prevention of graftversus-host disease (GvHD), which remains an obstacle to the successful outcome of allogeneic hematopoietic stem cell transplantation. However, Treg only constitute 1e5% of total nucleated cells in cord blood (CB) (90% (0.5e3.0 106 cells) (Figure 2A). The “negative” fraction containing CD25 cells was defined as “Tcon.” For the purpose of expansion, the whole of the positive fraction (Treg) was used. For Tcon, where appropriate, only a part (5 106 cells) of the negative fraction was cultured. Ex vivo culture was initiated with CD25þ cells at a concentration of 1 106 cells/mL in medium supplemented (as previously described) with IL-2 at 200 IU/mL, in the continued presence of CD3/28 beads, with an initial ratio of three Dynabeads:one cell. The cell numbers were assessed on an everyother-day basis and maintained at a concentration of
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Figure 2. Expanded CB Treg exert suppressive function. (A) Surface phenotype of CD25 positively and negatively selected CB T cells. Freshly isolated, CB-derived CD25þ and CD25neg fractions were analyzed at day 0, before expansion. (B) Treg phenotype. Ex vivoeexpanded CB cells after 14 days of culture were confirmed as CD4þ25þFOXP3þ127lo. (C) Expanded Treg show FOXP3 demethylation. Bisulfite sequencing in ex vivoeexpanded UCB-derived Treg showed significant DNA demethylation of the FOXP3 gene locus at amplicons 7, 9, 10 and 11 (11) when compared with Tcon. X-axis denotes the CpG island locus; Y-axis denotes percent methylation. (n ¼ 10). (D) Distribution of TCR repertoire is preserved in ex vivoeexpanded Treg: TCR repertoire remains preserved after expansion. Representative TCR repertoire distribution at day 14 of ex vivo expansion is shown. (E) Ex vivoeexpanded, CB-derived Treg significantly suppress immune response in allogeneic MLR. PBMCs from two donors were cultured together to generate MLR (D1 þ D2). Addition of Treg to the donor mixture (D1 þ D2) at a ratio of 1:1 significantly suppressed MLR. Y-axis denotes counts per minute (mean standard error of the mean, n ¼ 10). (F) Additive effect of p38MAPK inhibition. Pharmacologic inhibition of p38 MAP kinase (SB203580, 1 mmol/L) led to additive effect on CB Tregemediated MLR suppression (mean standard error of the mean, n ¼ 2).
Third-party umbilical CBederived regulatory T cells prevent xenogenic GvHD
95
Figure 3. Treg infusion prevents xenogeneic GvHD. (A) Circulating human lymphocytes. No effect of addition of CB Treg on day e1 was demonstrated on the circulating human lymphocytes in the PB of the xenogenic mouse model. (B) Condition of coat. Whereas PBMC recipients (left) show extensive loss of hair and skin erythema, recipients of Treg and PBMC (right) show an intact coat of fur. (C) Weight loss. Significantly greater weight loss was detected in the xenogeneic GvHD model in PBMC recipients after as little as 14 days after transplant when compared with recipients of Treg and PBMC (P ¼ 0.002; t test; n ¼ 20 mice in each arm). (D) GvHD score. Recipients of prophylactic Treg (107) demonstrated consistently lower GvHD scores on the basis of the Ferrara GvHD scale (P < 0.001; t test). An assessment of GvHD scoring was performed every 48e72 h (n ¼ 20 mice/group). (E) Histopathologic analysis. Formalin-fixed tissues were embedded in paraffin, and sections were stained with hematoxylin and eosin. Microscopic sections of lung from recipients of PBMC alone (left panel) show areas of evidence of GvHD in the form of apoptotic bodies, lymphocytic infiltration and loss of architecture in small intestine, liver and lung. Bone marrow aplasia and lymphocytic infiltration into the spleen was present. In contrast, microscopic evaluation of small intestine, liver and lung tissue from recipients of Treg and PBMC revealed preserved tissue histology. Preserved lymphoid follicles in spleen and hematopoiesis in bone marrow were observed. (F) Treg suppress levels of inflammatory cytokines in the xenogeneic GvHD mouse model. Measurements of circulating serum cytokines at day 14 showed a consistent decrease in the pro-inflammatory cytokine levels, including IL-6, interferon-g, interferon inducible protein-10, IL-5, macrophage inflammatory protein-1b and tumor necrosis factor-a in the Treg and PBMC recipients as compared with the PBMC-only recipients (n ¼ 5 mice/group; mean standard error of the mean). The unit of the Yaxis is pg/mL. (G) Overall survival. At a median follow-up of 30 days after PBMC infusion, the overall survival of PBMC recipients that also received Treg were >80% as compared with 90% of the final Treg product at 14 days of ex vivo culture were CD4þ25þFOXP3þ127lo (Figure 2B). By comparison,
